Facile F(ab′)2 Manufacturing: Strategies for the Production of Snake Antivenoms

Kumpalume, Peter; Boushaba, Rihab; Jones, R. L.; Slater, Nigel K.H.

doi:10.1205/09603080252938726

Cited by 7 publications

(15 citation statements)

References 22 publications

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“…The onset of F(ab′) 2 digestion occurring earlier when digesting prepurified IgG has been indicated in previous work by the results of the fluorescence quenching activity assay performed on digested samples of pure IgG and whole serum for different periods of digestion time (17). First, by increasing the initial IgG concentration in whole serum samples, more albumin and other serum proteins are introduced into the system.…”

Section: Resultsmentioning

confidence: 82%

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Kinetics of Whole Serum and Prepurified IgG Digestion by Pepsin for F(ab')2 Manufacture

Boushaba¹,

Kumpalume²,

Slater³

2008

Biotechnol Progress

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An alternative route for the production of polyclonal F(ab')(2) fragments that might be adopted for the facile preparation of antivenoms is assessed in this work. The method involves the digestion of whole serum by free pepsin, which results in reduction of the number of processing steps commonly in use, because it avoids the initial purification of IgG's prior to their proteolytic cleavage by the enzyme. Digestion kinetics of whole serum and caprylic acid prepurified IgG using free pepsin were monitored with SDS-PAGE followed by densitometric analysis and antigen binding activity assay of the digested samples. It was observed that with equal units of pepsin activity, caprylic acid prepurified IgG was digested more rapidly than whole serum but that the overall retention of antigen binding activity was significantly greater in the latter case. The estimated first-order digestion rate parameters were 11.8 and 4.42 microM min(-)(1) for pure IgG and whole serum, respectively. The K(m) value obtained for whole serum digestion was 33 microM and that for pure IgG digestion was 43.5 microM. Calibration with undigested whole serum and pure IgG samples of known concentrations was performed using SDS-PAGE followed by image analysis. A linear relationship was observed between the protein concentration and the respective band intensity within the range of concentrations investigated (0.63-31.2 microM IgG concentration). This technique proved to be relatively rapid, reproducible, and more precise than size-exclusion chromatography as a result of its F(ab')(2)/IgG resolving power. Staining and destaining protocols were reproduced in terms of staining and destaining times, volumes added, and compositions. Furthermore, all digestion experiments were performed in duplicate sets to monitor the extent of variation of the digestion kinetic parameters measured by this method. The results obtained from this technique confirm and quantify previous observations that pepsin digestion of whole serum is slower and easier to control than digestion of pure IgG and results in higher recovery of antigenic binding activity.

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Section: Resultsmentioning

confidence: 82%

“…The results of this assay are described elsewhere (17). Antigenic binding activity was monitored by the fluorescence quenching assay.…”

Section: Methodsmentioning

confidence: 99%

Kinetics of Whole Serum and Prepurified IgG Digestion by Pepsin for F(ab')2 Manufacture

Boushaba¹,

Kumpalume²,

Slater³

2008

Biotechnol Progress

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“…This is schematically illustrated in, Figure 4 (panels a and b) and has been confirmed experimentally. Antibodies within crude serum were shown to be more resistant to enzymatic digestion by pepsin than pure IgG preparations due to the presence of enzymatically favorable substrates, such as albumin, which could reduce by more than half the rate of IgG digestion (Boushaba et al, 2003;Jones & Landon, 2002;Jones & Landon, 2003;Kumpalume et al, 2002). In contrast, the trypsin digestion of native albumin in vitro was shown to be much more limited and provide little protection to the breakdown of IgG (Jones & Landon, 2002).…”

Section: Decoy Proteins To Protect Antibody Within the Gastrointestinmentioning

confidence: 99%

Targeted localized use of therapeutic antibodies: a review of non-systemic, topical and oral applications

Jones

Martino

2015

Critical Reviews in Biotechnology

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Therapeutic antibodies provide important tools in the "medicine chest" of today's clinician for the treatment of a range of disorders. Typically monoclonal or polyclonal antibodies are administered in large doses, either directly or indirectly into the circulation, via a systemic route which is well suited for disseminated ailments. Diseases confined within a specific localized tissue, however, may be treated more effectively and at reduced cost by a delivery system which targets directly the affected area. To explore the advantages of the local administration of antibodies, we reviewed current alternative, non-systemic delivery approaches which are in clinical use, being trialed or developed. These less conventional approaches comprise: (a) local injections, (b) topical and (c) peroral administration routes. Local delivery includes intra-ocular injections into the vitreal humor (i.e. Ranibizumab for age-related macular degeneration), subconjunctival injections (e.g. Bevacizumab for corneal neovascularization), intra-articular joint injections (i.e. anti-TNF alpha antibody for persistent inflammatory monoarthritis) and intratumoral or peritumoral injections (e.g. Ipilimumab for cancer). A range of other strategies, such as the local use of antibacterial antibodies, are also presented. Local injections of antibodies utilize doses which range from 1/10th to 1/100th of the required systemic dose therefore reducing both side-effects and treatment costs. In addition, any therapeutic antibody escaping from the local site of disease into the systemic circulation is immediately diluted within the large blood volume, further lowering the potential for unwanted effects. Needle-free topical application routes become an option when the condition is restricted locally to an external surface. The topical route may potentially be utilized in the form of eye drops for infections or corneal neovascularization or be applied to diseased skin for psoriasis, dermatitis, pyoderma gangrenosum, antibiotic resistant bacterial infections or ulcerated wounds. Diseases confined to the gastrointestinal tract can be targeted directly by applying antibody via the injection-free peroral route. The gastrointestinal tract is unusual in that its natural immuno-tolerant nature ensures the long-term safety of repeatedly ingesting heterologous antiserum or antibody materials. Without the stringent regulatory, purity and clean room requirements of manufacturing parenteral (injectable) antibodies, production costs are minimal, with the potential for more direct low-cost targeting of gastrointestinal diseases, especially with those caused by problematic antibiotic resistant or toxigenic bacteria (e.g. Clostridium difficile, Helicobacter pylori), viruses (e.g. rotavirus, norovirus) or inflammatory bowel disease (e.g. ulcerative colitis, Crohn's disease). Use of the oral route has previously been hindered by excessive antibody digestion within the gastrointestinal tract; however, this limitation may be overcome by intelligently applying one or more strate...

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“…1) are currently the preferred and often the only therapeutic choice for selected acute medical emergencies to eliminate complex, poorly characterized mixtures of target antigens. Purified polyclonal antibodies are often digested using specific proteases such as papain [9] or pepsin [10], and the specific antigen binding fragments (Fabs) further purified to remove the Fc fragment and other impurities and contaminants. CroFab TM , used for the treatment of crotalidae (rattlesnake) envenomation, the predominant venomous snakebite in the United States, has dramatically changed snakebite management since its release in December 2000 [21].…”

Section: Hyperimmune (Pab) Polyclonal Therapeuticsmentioning

confidence: 99%

“…As hyper-immunised serum contains a mixed population of antibodies against multiple epitopes, polyclonal antibody preparations are often considered more efficacious (especially for the treatment of acute illness and medical emergencies) as they can bind and neutralize multiple epitopes on the disease-causing agent. To minimize antigenicity against the species-specific Fc region, hyperimmune antibodies derived from animals are often digested with proteases such as papain [9] or pepsin [10] and the antigen binding fragments (Fabs) purified by chromatography [11]. The generation of monovalent Fab or divalent F(ab) 2 fragments significantly reduces hypersensitivity reactions and enhances product safety.…”

Section: Introductionmentioning

confidence: 99%